Colorectal carcinomas with KRAS codon 12 mutation are associated with more advanced tumor stages

KRAS mutation occurs in 35%-40% of colorectal cancer (CRC). The aim of our study was to evaluate the pathological and molecular features of specific KRAS mutated colorectal carcinomas. KRAS and BRAFV600E mutation tests were performed in 762 primary tumors from a consecutive cohort study of Chinese CRC patients.

Li et al BMC Cancer (2015) 15:340 DOI 10.1186/s12885-015-1345-3 RESEARCH ARTICLE Open Access Colorectal carcinomas with KRAS codon 12 mutation are associated with more advanced tumor stages Wenbin Li†, Tian Qiu†, Wenxue Zhi, Susheng Shi, Shuangmei Zou, Yun Ling, Ling Shan, Jianming Ying* and Ning Lu Abstract Background: KRAS mutation occurs in 35%-40% of colorectal cancer (CRC) The aim of our study was to evaluate the pathological and molecular features of specific KRAS mutated colorectal carcinomas KRAS and BRAFV600E mutation tests were performed in 762 primary tumors from a consecutive cohort study of Chinese CRC patients Methods: DNA mismatch repair (MMR) status was determined by immunohistochemistry (IHC) staining Assessment of KRAS and BRAF V600E mutational status was performed using a multiplex allele-specific PCR-based assay Results: Mutations of KRAS (34.8%) and BRAFV600E (3.1%) were nearly mutually exclusive Both KRAS- and BRAF- mutated tumors were more likely to be located at proximal colon than wild-type (WT) carcinomas KRAS-mutated carcinomas were more frequently observed in female patients (47.5% vs 37.1%, p = 0.005) and mucinous differentiation (34.7% vs 24.8%, p = 0.004), but have no difference between lymph node (LN) metastases and among pTNM stages Whereas, BRAF-mutated carcinomas more frequently demonstrated histologic features such as proximal location (60.9% vs 20.9%, p = 0.001), low-grade histology (43.5% vs 18.0%, p = 0.005), mucinous differentiation (69.6% vs 25.9%, p = 0.001) and deficient MMR (dMMR) (21.7% vs 7.6%, p = 0.03) In particular, KRAS codon 12 mutated carcinomas had increased lymph node metastasis (odds ratio [OR] = 1.31; 95% confidence interval [CI] = 1.04 to 1.65; P = 0.02) and were more likely in higher disease stage (III-IV) than that of WT carcinomas (OR = 1.30; 95% CI = 1.03 to 1.64; P = 0.03) However, there were no significant differences in lymph node metastasis and disease stage between KRAS codon 13 mutated carcinoma and WT carcinoma patients Conclusions: In summary, KRAS codon 12 mutation, but not codon 13 mutation, is associated with lymph node metastasis and higher tumor stages Keywords: KRAS mutation, BRAF mutation, Colorectal cancer, Codon 12 and 13, DNA mismatch repair Background Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer mortality in China [1] CRC is a multistep process based on the accumulation of somatic mutations and can be divided into at least two different and seemingly independent pathways, which is the chromosomal instability (CIN) and microsatellite instability (MSI) pathways [2-5] CIN occurs in about 85% patients with sporadic CRC and is thought to originate from a relatively uniform and linear * Correspondence: jmying@hotmail.com † Equal contributors Department of Pathology, Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, National Cancer Center, Beijing, China accumulation of genetic changes in APC, KRAS and TP53 genes [6] However, sporadic tumors with MSIhigh (MSI-H) are originated from promoter hypermethylation of the MLH1 gene, more frequently found in females, and tend to be poorly differentiated tumors, of mucinous subtype and often harboring somatic mutations in BRAFV600E [7,8] Dysfunction of the DNA mismatch repair (MMR) system is the main cause of MSI, which leads to accelerated accumulation of single nucleotide mutations and alterations in the length of simple, repetitive microsatellite sequences [9] Recently, MMR status, KRAS and BRAF mutation status have attracted remarkable attention due to their potential prognostic and predictive role in colorectal carcinomas © 2015 Li et al.; licensee BioMed Central This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Li et al BMC Cancer (2015) 15:340 [10-12] KRAS mutations are present in approximately 35% to 40% of colon cancers, with roughly 2/3 of these mutations in codon 12 and 1/3 in codon 13 [11,13] The presence of a KRAS mutation is predictive for resistance to anti-EFGR monoclonal antibodies (mAbs) in advanced colon cancer [13-16] However, the biological and functional consequences of KRAS mutations at codon 12 may be different from those at codon 13 [17-19] It has been suggested that patients whose tumors harbor a KRAS Gly13Asp mutation may benefit from anti-EGFR mAb therapy [20-22] The clinical significance of KRAS mutation in colorectal carcinoma patients is controversial; some studies reported no association with survival, whereas others suggested that patients with KRAS mutated colorectal carcinoma have poorer outcome for any mutation subtype, mutation in codon 12 only or codon 13 only [19,21,23,24] On the other hand, BRAF is also involved in the MAPK/ERK signaling pathway and oncogenic mutations in this gene have been identified in CRC [25] Several studies have reported a range of frequencies regarding BRAF mutations in colon cancer (7.1%–13.3%), with the most frequent mutation being a single substitution at nucleotide 1,799, substituting valine for glutamic acid (V600E) [7] Mutations in BRAF are most commonly found in microsatellite-unstable (MSI) tumors, whereas they are less common in microsatellite-stable (MSS) tumors [10,26,27] Mutations in KRAS and BRAF genes seem to occur in a mutually exclusive manner, and both are suggested as integral components for an effective molecular classification of colorectal cancer More accurate prediction of outcome among patients with CRC remains a worthy area of investigation Although the roles of MMR status, KRAS and BRAF mutations on clinical outcome are frequently documented, the accurate analysis of these features on clinicopathologic and prognosis with emphasis on the specific KRAS gene mutation is still missing The aim of this study was to evaluate the prognostic role of MMR status, BRAF mutations and specific KRAS point mutation in 762 patients in Chinese population, and several clinicopathologic features to better stratify colorectal cancer patients Methods Study population The clinicopathological records of 762 patients with corresponding paraffin-embedded material available for molecular analysis were retrospectively collected from the Department of Pathology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences, Beijing, China from December 2011 to December 2012 Patients who had a history of preoperative radiochemotherapy or gastrointestinal surgical resection were excluded Histopathological criteria were reviewed and included tumor diameter, pT and pN classification, grade of differentiation, Page of histological subtype, tumor location, tumor size as well as the pTNM stage The size of each tumor was evaluated by measuring its maximum diameter Grading was determined according to the 2010 WHO histological classification The pTNM stage system of the 7th edition AJCC cancer staging was used Evaluating of M stage was mainly according to confirmed pathological results and/or radiological data Location in the colon was designated as proximal colon for tumors located in the cecum, ascending colon and transverse colon, and as distal colon for tumors in the descending colon and sigmoid colon Mucinous differentiation in the tumor was defined by the presence of pools of extracellular mucin-containing clusters of carcinomatous cells When >50% of analyzed tumor demonstrated mucinous differentiation, the tumor was classified as mucinous carcinoma The study was approved by the Institute Review Board of the Cancer Hospital, CICAMS Each participant signed an Institutional Review Board approved informed consent in accordance with current guidelines KRAS and BRAFV600E mutation analysis Assessment of KRAS and BRAF V600E mutational status was performed in the Molecular Pathology Laboratory of Department of Pathology, CICAMS, using appropriate quality control procedures Mutation status was determined using genomic DNA extracted from macrodissected formalin-fixed, paraffin-embedded tumor tissue Both KRAS (codons 12 and 13) and BRAF (p.V600E) mutation tests were performed using a multiplex allele-specific PCR-based assay (ACCB, Beijing, China), together with the Stratagene Mx3000P (Agilent Technologies Inc, Santa Clara, CA), which assesses seven different potential mutations in KRAS codons 12 and 13 (Gly12Ala, Gly12Asp, Gly12Arg, Gly12Cys, Gly12Ser, Gly12Val, and Gly13Asp) Neither KRAS nor BRAFV600E mutated tumors were designated as WT carcinomas DNA mismatch repair proteins expression A panel of four-antibody of MMR proteins was performed as a routine practice in our pathological department, contained MLH1, PMS2, MSH2 and MSH6 All of the 762 samples were stained in an autostainer (Autostainer Link 48, Dako, Denmark) Primary mouse monoclonal antibodies included MLH1 antibody (ES05, Dako), MSH2 antibody (FE11, Dako) Primary rabbit monoclonal antibodies included MSH6 antibody (EP49, Dako) and PMS2 antibody (EP51, Dako) Carcinomas were considered as deficient MMR (dMMR) when there was a completely absent staining of a detectable nuclear signal in neoplastic cells for at least one protein While the adjacent normal mucosa or stromal/lymphoid cells that showed presence of nuclear staining are regarded as internal positive control Li et al BMC Cancer (2015) 15:340 Statistical analysis The primary objective of this study was to identify distinct clinicopathologic features associated with specific KRAS and BRAFV600E mutation status Differences of patient characteristics and clinicopathologic factors in the two-dimensional cross-comparison were evaluated statistically by Pearson’s χ2-test or Fischer’s exact test Statistical tests were two-sided, and P < 0.05 were considered significant Logistic regression models were used to detect associations of these characteristics with each of the specific KRAS mutations and provided estimates of odds ratio (ORs) and confidence intervals (CIs) Statistics were carried out using SPSS software (version 16.0 of SPSS, Chicago, IL, USA) Results Primary samples from 762 colorectal carcinoma patients were analyzed for KRAS, BRAF gene mutations and MMR status Mutations of KRAS occurred in 34.8% of colorectal carcinomas BRAFV600E mutation was demonstrated in 3.1% of colorectal carcinomas There was one tumor demonstrating mutations in both KRAS and BRAF; this case was excluded from the analysis Mutated carcinomas were compared with non mutated carcinomas for sex, age, histological features and molecular characteristics (Table 1) In addition, given that nonKRAS-mutated tumors include a distinct subset characterized by BRAF mutation, analyses were also performed to compare KRAS-mutated tumors with both BRAF-mutated tumors and the remaining subset of colorectal carcinomas, with observed neither somatic oncogene mutation Clinical information and morphological characteristics The mean age at presentation for KRAS-mutated carcinoma was 57.7 ± 11.3 years, which was no significantly different to that for non-mutated-KRAS carcinoma at 57.3 ± 11.5 years and WT carcinoma cases at 57.1 ± 11.5 years Furthermore, regarding the age, there was no significant difference in younger (